Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1989 Jul;86(13):4828–4832. doi: 10.1073/pnas.86.13.4828

Accessibility of receptor-bound urokinase to type-1 plasminogen activator inhibitor.

M V Cubellis 1, P Andreasen 1, P Ragno 1, M Mayer 1, K Danø 1, F Blasi 1
PMCID: PMC297508  PMID: 2544876

Abstract

Urokinase plasminogen activator (uPA) interacts with a surface receptor and with specific inhibitors, such as plasminogen activator inhibitor type 1 (PAI-1). These interactions are mediated by two functionally independent domains of the molecule: the catalytic domain (at the carboxyl terminus) and the growth factor domain (at the amino terminus). We have now investigated whether PAI-1 can bind and inhibit receptor-bound uPA. Binding of 125I-labeled ATF (amino-terminal fragment of uPA) to human U937 monocyte-like cells can be competed for by uPA-PAI-1 complexes, but not by PAI-1 alone. Performed 125I-labeled uPA-PAI-1 complexes can bind to uPA receptor with the same binding specificity as uPA. PAI-1 also binds to, and inhibits the activity of, receptor-bound uPA in U937 cells, as shown in U937 cells by a caseinolytic plaque assay. Plasminogen activator activity of these cells is dependent on exogenous uPA, is competed for by receptor-binding diisopropyl fluorophosphate-treated uPA, and is inhibited by the addition of PAI-1. In conclusion, in U937 cells the binding to the receptor does not shield uPA from the action of PAI-1. The possibility that in adherent cells a different localization of PAI-1 and uPA leads to protection of uPA from PAI-1 is to be considered.

Full text

PDF
4828

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Andreasen P. A., Nielsen L. S., Kristensen P., Grøndahl-Hansen J., Skriver L., Danø K. Plasminogen activator inhibitor from human fibrosarcoma cells binds urokinase-type plasminogen activator, but not its proenzyme. J Biol Chem. 1986 Jun 15;261(17):7644–7651. [PubMed] [Google Scholar]
  2. Andreasen P. A., Riccio A., Welinder K. G., Douglas R., Sartorio R., Nielsen L. S., Oppenheimer C., Blasi F., Danø K. Plasminogen activator inhibitor type-1: reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing. FEBS Lett. 1986 Dec 15;209(2):213–218. doi: 10.1016/0014-5793(86)81113-9. [DOI] [PubMed] [Google Scholar]
  3. Appella E., Robinson E. A., Ullrich S. J., Stoppelli M. P., Corti A., Cassani G., Blasi F. The receptor-binding sequence of urokinase. A biological function for the growth-factor module of proteases. J Biol Chem. 1987 Apr 5;262(10):4437–4440. [PubMed] [Google Scholar]
  4. Baker J. B., Low D. A., Simmer R. L., Cunningham D. D. Protease-nexin: a cellular component that links thrombin and plasminogen activator and mediates their binding to cells. Cell. 1980 Aug;21(1):37–45. doi: 10.1016/0092-8674(80)90112-9. [DOI] [PubMed] [Google Scholar]
  5. Bergman B. L., Scott R. W., Bajpai A., Watts S., Baker J. B. Inhibition of tumor-cell-mediated extracellular matrix destruction by a fibroblast proteinase inhibitor, protease nexin I. Proc Natl Acad Sci U S A. 1986 Feb;83(4):996–1000. doi: 10.1073/pnas.83.4.996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blasi F., Vassalli J. D., Danø K. Urokinase-type plasminogen activator: proenzyme, receptor, and inhibitors. J Cell Biol. 1987 Apr;104(4):801–804. doi: 10.1083/jcb.104.4.801. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chapman H. A., Jr, Vavrin Z., Hibbs J. B., Jr Macrophage fibrinolytic activity: identification of two pathways of plasmin formation by intact cells and of a plasminogen activator inhibitor. Cell. 1982 Mar;28(3):653–662. doi: 10.1016/0092-8674(82)90220-3. [DOI] [PubMed] [Google Scholar]
  8. Cubellis M. V., Nolli M. L., Cassani G., Blasi F. Binding of single-chain prourokinase to the urokinase receptor of human U937 cells. J Biol Chem. 1986 Dec 5;261(34):15819–15822. [PubMed] [Google Scholar]
  9. Danø K., Andreasen P. A., Grøndahl-Hansen J., Kristensen P., Nielsen L. S., Skriver L. Plasminogen activators, tissue degradation, and cancer. Adv Cancer Res. 1985;44:139–266. doi: 10.1016/s0065-230x(08)60028-7. [DOI] [PubMed] [Google Scholar]
  10. Fabricant R. N., De Larco J. E., Todaro G. J. Nerve growth factor receptors on human melanoma cells in culture. Proc Natl Acad Sci U S A. 1977 Feb;74(2):565–569. doi: 10.1073/pnas.74.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Genton C., Kruithof E. K., Schleuning W. D. Phorbol ester induces the biosynthesis of glycosylated and nonglycosylated plasminogen activator inhibitor 2 in high excess over urokinase-type plasminogen activator in human U-937 lymphoma cells. J Cell Biol. 1987 Mar;104(3):705–712. doi: 10.1083/jcb.104.3.705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ginsburg D., Zeheb R., Yang A. Y., Rafferty U. M., Andreasen P. A., Nielsen L., Dano K., Lebo R. V., Gelehrter T. D. cDNA cloning of human plasminogen activator-inhibitor from endothelial cells. J Clin Invest. 1986 Dec;78(6):1673–1680. doi: 10.1172/JCI112761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Goldberg A. R. Increased protease levels in transformed cells: a casein overlay assay for the detection of plasminogen activator production. Cell. 1974 Jun;2(2):95–102. doi: 10.1016/0092-8674(74)90097-x. [DOI] [PubMed] [Google Scholar]
  14. Granelli-Piperno A., Reich E. A study of proteases and protease-inhibitor complexes in biological fluids. J Exp Med. 1978 Jul 1;148(1):223–234. doi: 10.1084/jem.148.1.223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hajjar K. A., Hamel N. M., Harpel P. C., Nachman R. L. Binding of tissue plasminogen activator to cultured human endothelial cells. J Clin Invest. 1987 Dec;80(6):1712–1719. doi: 10.1172/JCI113262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hearing V. J., Law L. W., Corti A., Appella E., Blasi F. Modulation of metastatic potential by cell surface urokinase of murine melanoma cells. Cancer Res. 1988 Mar 1;48(5):1270–1278. [PubMed] [Google Scholar]
  17. Hekman C. M., Loskutoff D. J. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem. 1985 Sep 25;260(21):11581–11587. [PubMed] [Google Scholar]
  18. Holmberg L., Bladh B., Astedt B. Purification of urokinase by affinity chromatography. Biochim Biophys Acta. 1976 Aug 12;445(1):215–222. doi: 10.1016/0005-2744(76)90174-1. [DOI] [PubMed] [Google Scholar]
  19. Hébert C. A., Baker J. B. Linkage of extracellular plasminogen activator to the fibroblast cytoskeleton: colocalization of cell surface urokinase with vinculin. J Cell Biol. 1988 Apr;106(4):1241–1247. doi: 10.1083/jcb.106.4.1241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jones P., Benedict W., Strickland S., Reich E. Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells. Cell. 1975 Jul;5(3):323–329. doi: 10.1016/0092-8674(75)90108-7. [DOI] [PubMed] [Google Scholar]
  21. Klinger K. W., Winqvist R., Riccio A., Andreasen P. A., Sartorio R., Nielsen L. S., Stuart N., Stanislovitis P., Watkins P., Douglas R. Plasminogen activator inhibitor type 1 gene is located at region q21.3-q22 of chromosome 7 and genetically linked with cystic fibrosis. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8548–8552. doi: 10.1073/pnas.84.23.8548. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Leung K. C., Byatt J. A., Stephens R. W. The resistance of fibrin-stimulated tissue plasminogen activator to inactivation by a class PAI-2 inhibitor (minactivin). Thromb Res. 1987 Jun 15;46(6):755–766. doi: 10.1016/0049-3848(87)90068-5. [DOI] [PubMed] [Google Scholar]
  24. Loskutoff D. J., Linders M., Keijer J., Veerman H., van Heerikhuizen H., Pannekoek H. Structure of the human plasminogen activator inhibitor 1 gene: nonrandom distribution of introns. Biochemistry. 1987 Jun 30;26(13):3763–3768. doi: 10.1021/bi00387a004. [DOI] [PubMed] [Google Scholar]
  25. Lund L. R., Georg B., Nielsen L. S., Mayer M., Danø K., Andreasen P. A. Plasminogen activator inhibitor type 1: cell-specific and differentiation-induced expression and regulation in human cell lines, as determined by enzyme-linked immunosorbent assay. Mol Cell Endocrinol. 1988 Nov;60(1):43–53. doi: 10.1016/0303-7207(88)90118-9. [DOI] [PubMed] [Google Scholar]
  26. Mignatti P., Robbins E., Rifkin D. B. Tumor invasion through the human amniotic membrane: requirement for a proteinase cascade. Cell. 1986 Nov 21;47(4):487–498. doi: 10.1016/0092-8674(86)90613-6. [DOI] [PubMed] [Google Scholar]
  27. Nielsen L. S., Andreasen P. A., Grøndahl-Hansen J., Huang J. Y., Kristensen P., Danø K. Monoclonal antibodies to human 54,000 molecular weight plasminogen activator inhibitor from fibrosarcoma cells--inhibitor neutralization and one-step affinity purification. Thromb Haemost. 1986 Apr 30;55(2):206–212. [PubMed] [Google Scholar]
  28. Nielsen L. S., Kellerman G. M., Behrendt N., Picone R., Danø K., Blasi F. A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification. J Biol Chem. 1988 Feb 15;263(5):2358–2363. [PubMed] [Google Scholar]
  29. Ny T., Sawdey M., Lawrence D., Millan J. L., Loskutoff D. J. Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor. Proc Natl Acad Sci U S A. 1986 Sep;83(18):6776–6780. doi: 10.1073/pnas.83.18.6776. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Ossowski L., Biegel D., Reich E. Mammary plasminogen activator: correlation with involution, hormonal modulation and comparison between normal and neoplastic tissue. Cell. 1979 Apr;16(4):929–940. doi: 10.1016/0092-8674(79)90108-9. [DOI] [PubMed] [Google Scholar]
  31. Ossowski L. Plasminogen activator dependent pathways in the dissemination of human tumor cells in the chick embryo. Cell. 1988 Feb 12;52(3):321–328. doi: 10.1016/s0092-8674(88)80025-4. [DOI] [PubMed] [Google Scholar]
  32. Ossowski L., Reich E. Antibodies to plasminogen activator inhibit human tumor metastasis. Cell. 1983 Dec;35(3 Pt 2):611–619. doi: 10.1016/0092-8674(83)90093-4. [DOI] [PubMed] [Google Scholar]
  33. Pannekoek H., Veerman H., Lambers H., Diergaarde P., Verweij C. L., van Zonneveld A. J., van Mourik J. A. Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family. EMBO J. 1986 Oct;5(10):2539–2544. doi: 10.1002/j.1460-2075.1986.tb04532.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Petersen L. C., Lund L. R., Nielsen L. S., Danø K., Skriver L. One-chain urokinase-type plasminogen activator from human sarcoma cells is a proenzyme with little or no intrinsic activity. J Biol Chem. 1988 Aug 15;263(23):11189–11195. [PubMed] [Google Scholar]
  35. Picone R., Kajtaniak E. L., Nielsen L. S., Behrendt N., Mastronicola M. R., Cubellis M. V., Stoppelli M. P., Pedersen S., Danø K., Blasi F. Regulation of urokinase receptors in monocytelike U937 cells by phorbol ester phorbol myristate acetate. J Cell Biol. 1989 Feb;108(2):693–702. doi: 10.1083/jcb.108.2.693. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Plow E. F., Freaney D. E., Plescia J., Miles L. A. The plasminogen system and cell surfaces: evidence for plasminogen and urokinase receptors on the same cell type. J Cell Biol. 1986 Dec;103(6 Pt 1):2411–2420. doi: 10.1083/jcb.103.6.2411. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Pöllänen J., Hedman K., Nielsen L. S., Danø K., Vaheri A. Ultrastructural localization of plasma membrane-associated urokinase-type plasminogen activator at focal contacts. J Cell Biol. 1988 Jan;106(1):87–95. doi: 10.1083/jcb.106.1.87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Pöllänen J., Saksela O., Salonen E. M., Andreasen P., Nielsen L., Danø K., Vaheri A. Distinct localizations of urokinase-type plasminogen activator and its type 1 inhibitor under cultured human fibroblasts and sarcoma cells. J Cell Biol. 1987 Apr;104(4):1085–1096. doi: 10.1083/jcb.104.4.1085. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sprengers E. D., Kluft C. Plasminogen activator inhibitors. Blood. 1987 Feb;69(2):381–387. [PubMed] [Google Scholar]
  40. Stoppelli M. P., Corti A., Soffientini A., Cassani G., Blasi F., Assoian R. K. Differentiation-enhanced binding of the amino-terminal fragment of human urokinase plasminogen activator to a specific receptor on U937 monocytes. Proc Natl Acad Sci U S A. 1985 Aug;82(15):4939–4943. doi: 10.1073/pnas.82.15.4939. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Stoppelli M. P., Tacchetti C., Cubellis M. V., Corti A., Hearing V. J., Cassani G., Appella E., Blasi F. Autocrine saturation of pro-urokinase receptors on human A431 cells. Cell. 1986 Jun 6;45(5):675–684. doi: 10.1016/0092-8674(86)90782-8. [DOI] [PubMed] [Google Scholar]
  42. Sundström C., Nilsson K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937). Int J Cancer. 1976 May 15;17(5):565–577. doi: 10.1002/ijc.2910170504. [DOI] [PubMed] [Google Scholar]
  43. Thorsen S., Philips M., Selmer J., Lecander I., Astedt B. Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2. Eur J Biochem. 1988 Jul 15;175(1):33–39. doi: 10.1111/j.1432-1033.1988.tb14162.x. [DOI] [PubMed] [Google Scholar]
  44. Vassalli J. D., Baccino D., Belin D. A cellular binding site for the Mr 55,000 form of the human plasminogen activator, urokinase. J Cell Biol. 1985 Jan;100(1):86–92. doi: 10.1083/jcb.100.1.86. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES